Search

15 Pág. Departamento de Reproducción Animal​ (INIA), Extracellular vesicles (EVs) found in various biological fluids and particularly in reproductive fluids, have gained considerable attention for their possible role in cell- to- cell communication. Among, the different bioactive molecules cargos of EVs, MicroRNAs (miRNAs) are emerging as promising diagnostic biomarkers with high clinical potential. Aiming to understand the roles of EVs in bovine reproductive tract, we intended to characterize and profile the EVs of oviduct and uterine fluids (OF-EVs, UF-EVs) and their miRNA across the estrous cycle. Nanoparticle tracking analysis and transmission electron microscopy confirmed the existence of small EV population in OF and UF at all stages, (size between 30 and 200 nm; concentration: 3.4 × 1010 EVs/ml and 6.0 × 1010 EVs/ml for OF and UF, respectively, regardless of stage). The identification of EV markers (CD9, HSP70, and ALIX proteins) was confirmed by western blot. The miRNA analysis revealed the abundance of 310 and 351 miRNAs in OF-EVs and UF-EVs, respectively. Nine miRNAs were differentially abundant in OF-EVs between stages of the cycle, eight of them displayed a progressive increase from S1 to S4 (p, This work was supported by the Spanish Ministry of Science and Innovation (PID2019‐111641RB‐I00 to DR, and RTI2018‐093548‐B‐I00 to AGA); Minciencias‐Colombia (848‐2019) Postdoctoral Fellowship contract to KC‐B; Secretaría de Educación Superior, Ciencia y Tecnología e Innovación (SENESCYT‐Ecuador) to YNC; Sao Paulo Research Foundation, Brazil (FAPESP; #2017/20339‐3 and CNPq‐Brazil 304276/2018‐9 to CLVL; #2019/04981‐2 to RM; 2014/22887‐0 and 2015/21829‐9 to JCDS).

D-Pinitol (DPIN) is a natural occurring inositol capable of activating the insulin pathway in peripheral tissues, whereas this has not been thoroughly studied in the central nervous system. The present study assessed the potential regulatory effects of DPIN on the hypothalamic insulin signaling pathway. To this end we investigated the Phosphatidylinositol-3-kinase (PI3K)/Protein Kinase B (Akt) signaling cascade in a rat model following oral administration of DPIN. The PI3K/Akt-associated proteins were quantified by Western blot in terms of phosphorylation and total expression. Results indicate that the acute administration of DPIN induced time-dependent phosphorylation of PI3K/Akt and its related substrates within the hypothalamus, indicating an activation of the insulin signaling pathway. This profile is consistent with DPIN as an insulin sensitizer since we also found a decrease in the circulating concentration of this hormone. Overall, the present study shows the pharmacological action of DPIN in the hypothalamus through the PI3K/Akt pathway when giving in fasted animals. These findings suggest that DPIN might be a candidate to treat brain insulin-resistance associated disorders by activating insulin response beyond the insulin receptor., This work was supported by grants from the following institutions: Ministerio de Economía y Competitividad, Gobierno de España (Grant RTC-2016-4983-1), EU-ERDF-Instituto de Salud Carlos III (Grant PI19/01577), and Consejería de Economía, Conocimiento y Universidad, Junta de Andalucía (Grant P18-TP-5194), Proyecto de Investigación en Salud, Consejería de Salud y Familias (PI-0139-2018). D.M-V. (FI20/00227) holds a “PFIS” predoctoral contract from the National System of Health, EU-ERDF-Instituto de Salud Carlos III

In vitro culture can alter the development and quality of bovine embryos. Therefore, we aimed to evaluate whether nobiletin supplementation during EGA improves embryonic development and blastocyst quality and if it affects PI3K/AKT signaling pathway. In vitro zygotes were cultured in SOF + 5% FCS (Control) or supplemented with 5, 10 or 25 µM nobiletin (Nob5, Nob10, Nob25) or with 0.03% dimethyl-sulfoxide (CDMSO) during minor (2 to 8-cell stage; MNEGA) or major (8 to 16-cell stage; MJEGA) EGA phase. Blastocyst yield on Day 8 was higher in Nob5 (42.7 ± 1.0%) and Nob10 (44.4 ± 1.3%) for MNEGA phase and in Nob10 (61.0 ± 0.8%) for MJEGA phase compared to other groups. Mitochondrial activity was higher and lipid content was reduced in blastocysts produced with nobiletin, irrespective of EGA phase. The mRNA abundance of CDK2, H3-3B, H3-3A, GPX1, NFE2L2 and PPARα transcripts was increased in 8-cells, 16-cells and blastocysts from nobiletin groups. Immunofluorescence analysis revealed immunoreactive proteins for p-AKT forms (Thr308 and Ser473) in bovine blastocysts produced with nobiletin. In conclusion, nobiletin supplementation during EGA has a positive effect on preimplantation bovine embryonic development in vitro and corroborates on the quality improvement of the produced blastocysts which could be modulated by the activation of AKT signaling pathway., This work was funded by the Spanish Ministry of Science and Innovation (PID2019-111641RB-I00 to D.R. and RTI2018-093548-B-I00 to A.G.-A). Y.N.C. was supported by a predoctoral fellowship from the Secretaría Nacional de Educación Superior, Ciencia, Tecnología e Innovación (Convocatoria abierta 2017, SENESCYT-Ecuador). C.L.V.L. was supported by a BPE grant from Fundação de Amparo à Pesquisa do Estado de São Paulo, Brazil (FAPESP #2017/20339-3).